Team:ETH Zurich/MaterialMethods

From 2012.igem.org

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Contents

Material & Methods

Protocols

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Ligation

Ligation is performed with a ration of 1:5 plasmid backbone : Insert . The Ligation mix is incubated for at least 10 min at room temperature or for at least 1 hour at 16°C before the ligase is heat inactivated at 65°C for 20 min.





Ligation mix:

DNA Mix x µL
Ligase Buffer 10x 2.5 µL
Ligase 0.5 µL
H2O to 25 µL


Transformation

  • Allow competent cells to thaw on ice
  • Add ligation-mixture or 3-5 µl DNA to 50 µL cells
  • Incubate on ice for 30 min
  • Heatshock 45 sec at 42 °C
  • Add 900 µL LB medium and incubate in a shaker at 37 °C 60 min.
  • Spin cells down and remove supernatant
  • Resuspend cells in 50 µL medium and spread them onto a agar plate


Glycerol Stocks

For longtime storrage 0.6 mL of cells (overnight culture) are mixed with 0.4 mL 100 % Glycerol and stored at -80°C.

PCR

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Template 1 ng
Phusion buffer (5x) 10 µL
Forward Primer (10µM) 2.5 µL
Reverse Primer (10µM) 2.5 µL
Phusion polymerase 0.2 µL
H2O to 50 µL


For colony PCR the colony is resuspended in 10 µL water and the PCR mix is adjusted with respect to the additional 10 µL. The initial denaturation step is extended up to 5 min. PCR protocol:

  • Initial denaturation at 98°C for 30 sec
  • 25-35 cycles:
    • Denaturation at 98°C for 5 sec
    • Annealing for 20 sec (Temp. depends on Primer)
    • Extension at 72°C for 30 sec per kb
  • Final extension at 72 °C for 5 min

SDS-Page

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Compound 12% Running gel 5% Stacking gel
H2O 6.67 mL 3.54 mL
1.5 M Tris-HCl, pH 8.8 5 mL
0.5M Tris-HCl, pH 6.8 3 mL
10% (w/v) SDS 200 µL 80µL
Acrylamide/Bis-acrylamide (30%/0.8% w/v) 8 mL 1.32 mL
10%(w/v) APS 100 µL 50 µL
TEMED 30 µL 15 µL
Total 4 gels: 20 mL 8mL

As a marker for the SDS-Page “PageRuler plus prestained protein marker” is used. Gel is stained with Coomassie Blue.

Native-page acrylamide gels were made with the same composition, just lacking SDS and stacking layer. Also, native gels contained an acrylamide top to bottom gradient of 6 - 20 %.


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Cell Lysis

Lysis Buffer (Tris 50 mM, NaCl 150 mM, EDTA 5 mM, pH=7.4)

  • Spin down 1 mL of liquid culture
  • Resuspend in 1 mL of lysis buffer
  • Add lysozyme to a concentration of 1 mg/mL
  • Freeze cells in dry ice for 30 min
  • Thaw the cells and centrifuge at 4°C
  • Use supernatant for testing

Miller Assay

Z Buffer (NaH2PO4 40 mM, Na2HPO4 60 mM, KCl 10 mM, pH=7)

Add 4mg/mL of ortho-Nitrophenyl-β-Glactoside (ONPG) just before useage.

20 µL cell lysate is dissolved in 180 µL of Z-Buffer with ONPG. ONP activity is measured in a 96 wellplate at 420 nm every minute over a time period of 10min. β-Galactosidase activity determined based on the slope of the measurements.


TECAN plate reader Sample preparation

5 mL of fresh LB containing necessary antibiotic resistances was inoculated with 50 µL of overnight culture and grown at +37 °C while shaking vigorously. After cell density reaches OD600 ~ 0.1-0.2, 1 mL was withdrawn and mixed with IPTG. Then, three 200 µL samples were taken and transferred into sterile 96 well plate (Thermofisher scientific, Denmark) and used for data analysis as a triplicate. Cells were later grown in TECAN Infinite 200Pro plate reader (Switzerland) at 37 °C, while shaking in orbital mode with 5 mm amplitude. Each 15 min fluorescence (excitation at 488±4.5 nm, emission at 530±10 nm; 25 flashes and integration over 20 µs) and cell density (absorbance at 600±4.5 nm; 15 flashes) measurements were taken.

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Single Cell analysis using flow cytometry

For sample preparation the OD600 was measured to gauge the volume for sample-taking ensuring similar cell-concentrations for each measure. Cells are harvested and resuspended in an appropriate amount of Phosphate buffered saline (PBS). It is possible that the cells has to be diluted later because the flow is too high due to high concentration.

Measurements were performed in the BD LSRFortessaTM using following lasers and filters:

  • SSC and FSC: 488 nm - 488 nm/10 nm
  • GFP: 488 - 530/30
    • excitation: 488 nm
    • emission: 530 ± 15 nm
  • mRFP and mCherry: 561 - 610/20
  • YFP: 488 - 542/27
  • eCFP: 445 - 473/10
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High-performance liquid chromatography (HPLC)

The HPLC was performed with a ... column. Sample preparation: Cells were lysed in acteonitril incubated for 15 min on ice and then centrifuged for 30 min at top speed. The supernatant was applied onto the column.


HPLC Method:

  • Column (preheated up to 40 °C) was equilibrated with 92% A and 8% B
  • 50 µl per sample were applied to the column
  • The proportion B was increased linearly to 50% in 7 min. The last 3 min it is increased to 100%.

Buffers:

A: Water , 0.1% formic acid

B: Methanol, 0.1% formic acid

Purification of TetRDBD-UVR8-HisTag

Cells, expresing TetRDBD-dUVR8 with 7xHis tag at the C-terminus under Ptac promoter, were grown overnight in the 100 mL of LB media at 37 °C without IPTG induction. Culture were then centrifuged at 4000 rpm for 20 min at 4°, supernatant discarded and cells were resuspended at 10 mL of Lysis buffer (50 mM K-PO4, 500 mM NaCl, 10 mM Imidazol) and lysed with 1 mg/mL lysozyme at room temperature followed with rapid freezing in dry ice and kept frozen for 1h. Cells then were thawed and incubated for another hour with DNase and frozen as before. Cells debris were removed by centrifugation and supernatant was loaded on Ni ion affinity chromatography column, washed with lysis buffer containing 50 mM imidazol and eluted with 50 mM K-PO4, 500 mM NaCl, 300 mM imidazol. Elute was concentrated and diluted 1:1 with glycerol and stored at -20 °C.


Mediums

Agar plates

  • 1 % gels in TAE buffer

LB medium (1L)

  • 10 g Bacto-tryptone
  • 5 g yeast extract
  • 10g NaCl

LB agar (1L)

  • LB medium
  • 15 g Agar

Chemicals

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Antibiotic Stock Solutions (1000x)

  • Kanamycin: 50 mg/mL
  • Ampicillin: 100 mg/mL
  • Chloramphenicol: 34 mg/mL

X-Gal

  • 20 mg/mL in DMSO

IPTG

  • 100 mM in water

aTc

  • 1 mg/mL in ethanol




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